For genes encoding proteins that are required for organismal or cell viability, an inability to generate and examine loss-of-function mutant phenotypes can be an impediment to elucidating protein function. Homozygous mutant individuals may die at an early stage in development, making it difficult to establish the differen roles that the relevant proteins play at later stages of development. Tissue-specific generation of homozygous mutant clones, or expression of dsRNA, can in some cases overcome this limitation. However, these strategies are subject to the problem of protein perdurance beyond the time at which gene expression ceases, which can complicate analyses in which rapid elimination of gene function is required. Temperature-sensitive (ts) mutations provide an alternative means of conditionally eliminating gene product function. However, ts mutants are laborious to isolate and cannot be applied to the study of protein function in homeothermic organisms such as mammals. The objective of the investigations described in this proposal is the development of a general method for rapid, spatially- and temporally-controlled light-induced elimination of proteins of interest for phenotypic analysis.
Two specific aims will be pursued to develop and test two distinct experimental methods to achieve protein elimination.
Specific Aim 1 will utilize a small protein tag that we refer to as the photodegron, which can be expressed as a genetic fusion to other proteins. The photodegron undergoes a light-dependent conformational change that exposes a degradation signal recognized by a ubiquitous class of ubiquitin ligases, the N-recognins. In preliminary studies carried out in yeast, the photodegron mediated light-dependent elimination of function of the heterologous proteins to which it was attached. Continuing work will examine the kinetics of photodegron-induced degradation in yeast and test the ability of the photodegron to direct protein degradation in Drosophila embryos. Additional studies will increase the versatility of the photodegron method by incorporating a signal that targets it for degradation via the Nedd4 family of ubiquitin ligases. The investigations in Specifi Aim 2 will pursue a second strategy to achieve light- induced protein degradation in vivo by making use of the light-dependent interaction between the Arabidopsis thaliana proteins Cryptochrome 2 (CRY2) and CIB1. Planned experiments will test the ability of CRY2-ubiquitin ligase fusion proteins to mediate the degradation of proteins fused to CIB1 in Drosophila embryos. Based on recent advances in the understanding of light-responsive proteins and of ubiquitin/proteasome-mediated protein degradation, we hypothesize that it will be possible to use the sophisticated genetics of yeast and Drosophila to develop facile experimental strategies for rapid, temporally- and spatially-controlled protein elimination. These methods will overcome several limitations associated with currently available strategies for generating protein loss-of-function phenotypes and will provide a powerful tool for the study of medically important proteins involved in a wide variety of biological questions and experimental organisms.
Much of our understanding of biology has been built on the analysis of genetic mutations and their phenotypic consequences. However, for mutations that result in cell or organism death, it can be difficult to elucidate the normal roles that the affectd genes/proteins play in multiple tissues or at multiple times during the life of the organism. The goal of these proposed investigations is to develop a convenient, generally applicable strategy for rapid, temporally- and spatially-controlled elimination of proteins of biomedical interest via exposure to light. The utility of this technology in various cellular and organismal contexts will permit the analysis of the functions of many proteins that cannot be studied by more conventional mutational approaches.
